scholarly journals Characterization of strand exchange activity of yeast Rad51 protein.

1997 ◽  
Vol 17 (9) ◽  
pp. 5359-5368 ◽  
Author(s):  
E Namsaraev ◽  
P Berg
Keyword(s):  
Genetic Recombination ◽  
Strand Exchange ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Ssdna Binding Protein ◽  
Linear Dsdna ◽  
Circular Ssdna

The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.

scholarly journals Structure of a filament of stacked octamers of human DMC1 recombinase

2013 ◽  
Vol 69 (4) ◽  
pp. 382-386 ◽  
Author(s):  
Liqin Du ◽  
Yu Luo
Keyword(s):  
Electron Microscopy ◽  
Dna Sequences ◽  
Double Strand Breaks ◽  
Strand Exchange ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Double Stranded Dna ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Remote Homologs

Eukaryal DMC1 proteins play a central role in homologous recombination in meiosis by assembling at the sites of programmed DNA double-strand breaks and carrying out a search for allelic DNA sequences located on homologous chromatids. They are close homologs of eukaryal Rad51 and archaeal RadA proteins and are remote homologs of bacterial RecA proteins. These recombinases (also called DNA strand-exchange proteins) promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. An octameric form of a truncated human DMC1 devoid of its small N-terminal domain (residues 1–83) has been crystallized. The structure of the truncated DMC1 octamer is similar to that of the previously reported full-length DMC1 octamer, which has disordered N-terminal domains. In each protomer, only the ATP cap regions (Asp317–Glu323) show a noticeable conformational difference. The truncated DMC1 octamers further stack with alternate polarity into a filament. Similar filamentous assemblies of DMC1 have been observed to form on DNA by electron microscopy.

scholarly journals Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Chun Zhou ◽  
Sergei Pourmal ◽  
Nikola P Pavletich
Keyword(s):  
Double Strand Breaks ◽  
Helicase Domain ◽  
Dna Double Strand Breaks ◽  
Intact Mouse ◽  
Strand Breaks ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Dna Complex ◽  
Stalled Replication Forks ◽  
Tunnel Entrance

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5’ end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5’ but not 3’ end, explaining how Rpa regulates cleavage polarity.

scholarly journals Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2′-guanosine

2019 ◽  
Vol 47 (17) ◽  
pp. 9410-9422 ◽  
Author(s):  
Andrea M Kaminski ◽  
Kishore K Chiruvella ◽  
Dale A Ramsden ◽  
Thomas A Kunkel ◽  
Katarzyna Bebenek ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Steady State Kinetics ◽  
Ligase Iv ◽  
Template Dna ◽  
Dna Substrate

Abstract DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2′-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

scholarly journals Mutations in Caenorhabditis elegans him-19 Show Meiotic Defects That Worsen with Age

2010 ◽  
Vol 21 (6) ◽  
pp. 885-896 ◽  
Author(s):  
Lois Tang ◽  
Thomas Machacek ◽  
Yasmine M. Mamnun ◽  
Alexandra Penkner ◽  
Jiradet Gloggnitzer ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Male Meiosis ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Recombination Protein ◽  
High Incidence ◽  
Bivalent Formation ◽  
Synaptonemal Complex Formation

From a screen for meiotic Caenorhabditis elegans mutants based on high incidence of males, we identified a novel gene, him-19, with multiple functions in prophase of meiosis I. Mutant him-19(jf6) animals show a reduction in pairing of homologous chromosomes and subsequent bivalent formation. Consistently, synaptonemal complex formation is spatially restricted and possibly involves nonhomologous chromosomes. Also, foci of the recombination protein RAD-51 occur delayed or cease altogether. Ultimately, mutation of him-19 leads to chromosome missegregation and reduced offspring viability. The observed defects suggest that HIM-19 is important for both homology recognition and formation of meiotic DNA double-strand breaks. It therefore seems to be engaged in an early meiotic event, resembling in this respect the regulator kinase CHK-2. Most astonishingly, him-19(jf6) hermaphrodites display worsening of phenotypes with increasing age, whereas defects are more severe in female than in male meiosis. This finding is consistent with depletion of a him-19-dependent factor during the production of oocytes. Further characterization of him-19 could contribute to our understanding of age-dependent meiotic defects in humans.

scholarly journals RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization

2002 ◽  
Vol 184 (6) ◽  
pp. 1649-1660 ◽  
Author(s):  
Jong-Il Kim ◽  
Ajay K. Sharma ◽  
Stephen N. Abbott ◽  
Elizabeth A. Wood ◽  
David W. Dwyer ◽  
...  
Keyword(s):  
Deinococcus Radiodurans ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Exchange Reactions ◽  
Ssdna Binding ◽  
E Coli ◽  
Dna Strand Exchange ◽  
Ssdna Binding Protein ◽  
Dna Strand

ABSTRACT The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecADr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecADr protein binds much better to duplex DNA than the RecAEc protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

scholarly journals DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

2020 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Stephen Pu ◽  
Juncheng Wang ◽  
Dinshaw J. Patel ◽  
Scott Keeney
Keyword(s):  
Self Assembly ◽  
Chromosome Structure ◽  
Coiled Coil ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Breakage ◽  
And Control

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)—essential, conserved components of the DSB machinery. Each subcomplex of Rec114–Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114–Mei4–Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

scholarly journals Mechanism and Control of Meiotic DNA Double-Strand Break Formation in S. cerevisiae

2021 ◽  
Vol 9 ◽  
Author(s):  
Vikash Kumar Yadav ◽  
Corentin Claeys Bouuaert
Keyword(s):  
Strand Break ◽  
Current View ◽  
Dna Double Strand Breaks ◽  
Recombination Mechanism ◽  
Regulatory Pathways ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
And Function ◽  
And Control

Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.

scholarly journals Human Gene Targeting by Adeno-Associated Virus Vectors Is Enhanced by DNA Double-Strand Breaks

2003 ◽  
Vol 23 (10) ◽  
pp. 3550-3557 ◽  
Author(s):  
Daniel G. Miller ◽  
Lisa M. Petek ◽  
David W. Russell
Keyword(s):  
Gene Targeting ◽  
Human Gene ◽  
Retroviral Vectors ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Adeno Associated Virus ◽  
Strand Breaks ◽  
Double Stranded Dna ◽  
Linear Molecules

ABSTRACT The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.

scholarly journals Generation of Markerless Deletions in the Nosocomial PathogenClostridium difficileby Induction of DNA Double-Strand Breaks

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Elena-Stella Theophilou ◽  
Prerna Vohra ◽  
Maurice P. Gallagher ◽  
Ian R. Poxton ◽  
Garry W. Blakely
Keyword(s):  
Clostridium Difficile ◽  
Regulatory Elements ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Strand Breaks ◽  
Allelic Exchange ◽  
Genes Encoding

ABSTRACTClostridium difficileis an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions inC. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddABmutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of thefliCgene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCEMost sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution inClostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions inC. difficileand expands the toolbox available for genetic analysis of this important anaerobic pathogen.

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